Competitive Internal Amplification Controls for Nucleic Acid Sequencing
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Solution Overview
Problem
Next-generation sequencing (NGS) methods face challenges due to high intra-lab and inter-lab variation, primarily caused by non-systematic biases introduced during nucleic acid library preparation, which affects the accuracy and reproducibility of nucleic acid abundance measurements, limiting their clinical and diagnostic applications.
Innovation Solution
A method involving the use of competitive internal amplification controls (IACs) that share identical priming sites with native nucleic acid templates to control for PCR efficiency and reduce non-systematic errors, allowing for the normalization of nucleic acid representation and accurate measurement of copy numbers with reduced sequencing depth.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If next-generation sequencing is used to acquire comprehensive genetic information, then the accuracy and comprehensiveness of nucleic acid analysis is improved, but intra-lab and inter-lab variation increases due to non-systematic biases in library preparation
Solution Approach 1:
The patent introduces competitive internal amplification controls (IACs) as intermediary molecules that compete with native nucleic acid templates during PCR amplification. These IACs contain known concentrations and share identical priming sites with target sequences, serving as mediators to monitor and correct for non-systematic biases introduced during library preparation, thereby enabling accurate normalization across different laboratories
Solution Approach 2:
The methodology implements feedback control by measuring the proportion of sequencing reads corresponding to competitive IAC sequences versus native target sequences. This feedback information is used to calculate normalization factors that correct for technical variation in library preparation efficiency, allowing iterative optimization and standardization of sequencing results across multiple laboratories
2Measurement precision
If deep sequencing is performed to ensure sufficient reads for all transcripts, then measurement completeness is improved, but sequencing cost and time increase significantly
Solution Approach 1:
The patent performs preliminary action by adding competitive internal amplification controls with known concentrations to the nucleic acid sample before library preparation and sequencing. This pre-established reference system allows for post-sequencing normalization that accurately determines original transcript abundances without requiring excessive sequencing depth, thereby reducing both time and cost while maintaining measurement precision
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enhances the reproducibility and accuracy of nucleic acid abundance measurements across laboratories, reduces the need for deep sequencing, and lowers the cost of NGS by normalizing nucleic acid representation, enabling more efficient and cost-effective clinical diagnostics.
Implementation Method 1
competitive internal amplification controls (IACs) that share identical priming sites with native nucleic acid templates to control for PCR efficiency
Data Source
AI summary
Methods for standardized sequencing of nucleic acids and uses thereof are described. The identification of genetic information is becoming a key piece of information for the diagnosis and treatment of many diseases. In order to make such diagnostic tool readily available, it is desired that this identification be as efficient and as inexpensive as possible.